Sensitivity to ALK-Directed Therapy in Osteosarcoma With an Acquired ALK Rearrangement

Sarcomas are clinically, histologically, and molecularly heterogeneous, with at least 70 distinct subtypes occurring in bone and soft tissues. 1 Molecular alterations most commonly involve tumor suppressors (eg, cell cycle control genes, TP53 ), DNA damage response genes, epigenetic regulators, and, rarely, receptor tyrosine kinases (RTK). 2,3 The prevalence of a given class of alterations varies widely among histologic types. High-grade osteosarcoma is a malignant tumor of bone with diverse genomics, including mutations in TP53 , RB1 , FGFR1 , IGF1R, and DNA structural alterations. 4-7 In most sarcomas, including osteosarcoma, chemotherapy has traditionally been the backbone of therapy as there is an absence of classic kinase alterations as the main drivers (with exceptions such as GI stromal tumor [GIST], dermato ﬁ brosarcoma pro-tuberans, and in ﬂ ammatory myo ﬁ broblastic tumor [IMT]).

, followed by disease stabilization on subsequent scans (126.6% from baseline).After 5 months of alectinib, there was progression and he was transitioned to lorlatinib.Serial imaging confirmed stable disease for over 400 days (best response, stable disease [-11% by RECIST 1.1]; Fig 4).The patient continued lorlatinib with clinical benefit for 14 months, at which point he developed a wound infection in the right knee.This was managed with antibiotics and surgical debridement.The patient was admitted to his local hospital with sepsis.Despite maximal medical intervention, his health condition continued to decline.Ultimately, the decision was made to transition to comfort measures, and he passed away peacefully with his family at the bedside.

Histology
The primary right femur osteosarcoma was characterized by clusters and sheets of tumor cells associated with osteoid and woven bone, prototypical of a high-grade osteosarcoma.A thigh recurrence at 6 years displayed intermingling of conventional (osteoblastic), chondroblastic, giant cell-rich, and telangiectatic (cystic) areas, representing a recurrent high-grade osteosarcoma.Resection of a right upper lobe (RUL) metastasis at year 7 showed high-grade osteosarcoma with osteoid and woven bone.The resected metastases in the RML, right lower lobe (RLL), and diaphragm at year 8 displayed high-grade pleomorphic histology, with sheets of hyperchromatic tumor cells, extensive necrosis, and scattered multinucleated giant cells.Although there were no overt osteoid or woven bone deposition, that tumor was considered compatible with metastatic/recurrent osteosarcoma because of its histologic similarities to those in the previous specimens, and the subsequent molecular workup.Moreover, there were no distinctive histologic features in these specimens that would suggest to the pathologist to evaluate for an acquired ALK fusion event (Fig 3).

Molecular Genetic Profiling
The initial clinical molecular profiling was performed on the RML metastasis at year 8 after the primary diagnosis (Fig 2).Comprehensive genotyping using RNAseq showed an EML4:: ALK fusion (confirmed by immunohistochemistry [IHC] and florescence in situ hybridization [FISH]).In addition, there were TP53 G199V and FGFR1 M276I mutations, as well as CDKN2A loss by DNAseq, the latter also confirmed by FISH.The presence of ALK fusion was confirmed in the concurrent RLL and diaphragm metastases by IHC.The follow-up cytology specimen from RLL a year later had overlapping alterations of ALK, TP53, and CDKN2A, and lacked the FGFR1 mutation.
The primary tumor and thigh recurrence at 6 years were negative for ALK rearrangement by FISH or IHC (nextgeneration sequencing [NGS] failed because of decalcification).The RUL tumor with classic osteosarcoma morphology at year 7 had overlapping alterations with the previously described RML tumor, including TP53 and FGFR1 mutations, as well as CDKN2A loss, and it also did not harbor the ALK fusion (confirmed by RNAseq, FISH, and IHC).

Phylogenetic Analysis
Phylogenetic reconstruction of the tumor evolutionary history showed shared variants in multiple polyguanine repeats in independent samples from the primary knee tumor, thigh recurrence, and metastases to the lung and diaphragm, confirming that all tumors were clonally related.On the phylogenetic tree (Fig 5), samples segregated along two main branches: one branch comprised all the primary tumor samples (Kn1-9) and one region from the thigh recurrence (Th1); the other branch contained the second thigh recurrence region (Th2) and all thoracic metastases.These results are compatible with a scenario in which a localized subclone in the year 6 thigh recurrence seeded the lung metastases.Although the putative metastasis-seeding region of the thigh recurrence (Th2) and RUL metastasis (RUL1-3) at 7 years were negative for the ALK fusion, all other lung metastases (RML1-3, RLL1-2), as well as the diaphragm metastases at 8 years (Di1-3), were positive.This indicates a descendant of the putative metastatic subclone resident in Th2 acquired the ALK fusion, and that some metastases (RLL, RML, and Di) were seeded after this acquisition, while other metastases (RUL) were seeded independently (either earlier in time or by a parallel descendant of the Th2 subclone).

Autopsy Findings
Autopsy was performed after consent.There was a large RLL metastasis with extensive necrosis (>90%), along with inferior vena cava tumor thrombus.Microscopically, tumor cells appeared spindled-pleomorphic and showed diffuse strong ALK immunoreactivity, consistent with ALK-rearranged highgrade osteosarcoma.Bilateral patchy acute pneumonia and infection of right leg soft tissue were identified.The cause of death was metastatic high-grade osteosarcoma complicated by infection.
Methods are discussed in the Data Supplement (Appendix 1).

Discussion
Here, to our knowledge, we document the first known acquired ALK rearrangement in osteosarcoma.Reconstructing the timeline and integration of morphologic, molecular, and phylogenetic findings confirmed shared clonal origin between the primary and the recurrent/metastatic tumors despite the morphologic and molecular variety.The sensitivity to ALK-targeted therapy was demonstrated with prolonged disease stabilization of the previously rapidly progressing disease and extensive tumor necrosis in the autopsy.Finally, the changes in cfDNA allele fraction and emergence of a resistance mutation confirm antitumor pressure.Although this is a rare occurrence, our report highlights the potential for acquired kinase mutations in heavily treated malignancies and suggest ALK-directed therapy may be an option for acquired ALK rearrangements.
In the present case, the ALK rearrangement first emerged in lung metastases 8 years after resection of the primary.Phylogenetic analysis revealed that a subclone in the patient's thigh recurrence was the likely origin of the lung metastases.Although the patient received multiple cycles of chemotherapy and radiotherapy, interestingly, the first ALK-rearranged tumors developed in the setting of direct tumor exposure to microwave ablation and radiation, which may be suggestive of a thermal and/or radiation-related event or resistance mechanism.Emergence of ALK rearrangements in response to therapy has been reported in lung carcinomas 14,15 ; however, the chemotherapy/radiotherapy/ ablation status of these tumors was not clearly documented.To our knowledge, this phenomenon of an acquired ALK rearrangement in the setting of extensive therapies has not been documented in sarcomas.In addition, there is only a single patient with osteosarcoma in a previous case series of ALK-rearranged nonlung solid tumors. 16However, the context of primary versus acquired status and the oncogenic Case Report role of the ALK rearrangement are unclear on the basis of the data provided.Overall, to our knowledge, the findings of our case represent the first actionable ALK rearrangement in osteosarcomas, as well as its acquired status, which previously has not been established in sarcomas.
In addition to the molecular evolution of this osteosarcoma, there were morphologic changes over time.The resected RML metastasis, the first tumor where we detected the ALK rearrangement, was morphologically distinct (ie, an absence of osteoid formation) from the previous tumors, including an earlier lung metastasis with typical osteosarcoma morphology.This observation raised the possibility of a new primary tumor as a clinical consideration during the patient's care.This was ruled out by integrating phylogenetic assessment into the NGS findings (Figs 2 and 5).Although the phylogenetic evaluation is not a clinical assay, in the future, it may assist in understanding the temporal evaluation of genetic lesions, both for precursor lesions 17 and subsequent metastases. 18These findings highlight that relying on morphologic features alone would not be adequate to characterize tumors in scenarios such as this case.Overall, our data emphasize the importance of multisite sampling at different stages of disease progression while integrating molecular data to guide therapeutic considerations.This integrative approach will aid the pathology and oncology teams in determining if there is a second independent primary tumor and identify actionable alterations.
In conclusion, this ALK rearrangement in an osteosarcoma is a rare finding that demonstrates acquired RTK alterations are possible in sarcoma.This supports the concept of serial molecular testing through the course of treatment where targeted therapy may be a therapeutic option.Finally, phylogenetic analysis can provide valuable information on the clonal relationship between primary tumors and metastases for morphologically and molecularly challenging cases, which may have implications for treatment decisions.

FIG 1 .
FIG 1. Coronal computed tomography image of a lytic lesion centered in the medial femoral condyle corresponding to the primary osteosarcoma at presentation.Note the presence of a pathologic fracture.

FIG 2 .
FIG 2. Clinical timeline, tumor morphology, and molecular genotype.Note the lack of overt osteoid morphology coincides with emergence of the ALK rearrangement seen in the recent recurrences.The designations in parenthesis are for comparison to Figure 5. Chemo, chemotherapy; CTMA, CT microwave ablation; Di, diaphragm; empty box, not tested; Kn, knee; neg, negative; pos, positive; RadioTx, radiotherapy; RLL, right lower lobe; RML, right middle lobe; RUL, right upper lobe; Th, thigh.

FIG 3 .FIG 4 .
FIG 3. Representative histopathologic images are as follows: (A-D) thigh recurrence at 6 years (ALK rearrangement negative) showing (A) chondroblastic features with a chondroid matrix and variable cellularity, (B) telangiectatic features with lakes of blood admixed with malignant cells, (C) prototypical high-grade osteosarcoma with tumor cells associated with osteoid and woven bone, and (D) giant cell-rich morphologies.(E) Right upper lobe metastasis at 7 years (ALK rearrangement-negative) with prototypical high-grade osteosarcomatous morphology.(F) Right middle lobe metastasis at 8 years (ALK rearrangement positive) showing high-grade pleomorphic histology, with pleomorphic tumor cells, and scattered multinucleated giant cells without definitive osteoid or woven bone deposition.Although the thigh recurrence showed a variety of morphologies, the lymphovascular invasion showed the prototypical osteosarcoma morphology as depicted by (C).